Insulin resistance is linked to many of the most prevalent and devastating age-related pathologies, including Type 2 diabetes, cardiovascular disease and cognitive dysfunction. Skeletal muscle accounts for up to 85% of insulin-mediated blood glucose clearance, and glucose uptake (GU) is a rate-controlling step for muscle glucose metabolism. Calorie restriction (CR) enhances insulin-mediated GU in skeletal muscle from old rats and humans, but the cellular mechanisms are poorly understood. The broad, long-term goal is to advance understanding of mechanisms to improve insulin sensitivity, leading to healthy aging. The Specific Aims are: 1) To identify the specific mechanism that is responsible for AS160's role in the CR-induced improvement in insulin-mediated GU by skeletal muscle; 2) To discover novel, CR-responsive phosphoproteins that are insulin- regulated and Akt-dependent in skeletal muscle; 3) To determine the influence of CR on AMP-activated protein kinase (AMPK) heterotrimer-specific activity in skeletal muscle and to determine the extent to which an AMPK activating compound enhances CR's effect on insulin-mediated GU by muscle. Newly created AS160-null rats with adeno-associated virus-delivered wildtype or phosphomutated AS160 expression will be used to reveal if AS160 site-selective phosphorylation is essential for greater insulin-mediated GU with CR. Although Akt- dependent AS160 phosphorylation is important for insulin's full effect on GU, it is likely that other Akt- substrates also contribute to CR's effects on insulin sensitivity. However, none have been identified. Accordingly, we will use quantitative mass spectrometry-based phosphoproteomics to analyze muscles (from old AL vs. CR rats) treated insulin and selective Akt-inhibitor to discover novel protein phosphorylation sites regulated by CR, insulin and/or Akt. Based on the phosphoproteomics data, we will create genetically modified L6 muscle cells to test if these phosphoproteins control insulin-mediated GU. AMPK is a key intracellular energy sensor. Some studies have reported greater AMPK activation in muscles of CR animals, but others have not. These discrepancies may be in part because prior studies have only assessed total AMPK. AMPK is a heterotrimeric protein complex comprised of a catalytic subunit (?1 or ?2 isoform) and 2 regulatory subunits (?1 or ?2; and ?1, ?2 or ?3). Because AMPK?s diverse bioeffects depend on specific heterotrimers, we will resolve if CR effects are AMPK heterotrimer-selective. The level of CR in a typical rodent CR protocol (eating 60% of AL intake) is unrealistic for translation to humans. We will assess both a typical CR protocol (eating 60% of AL intake) and a more feasible protocol (eating 85% of AL intake). Because less severe CR may be less effective, and prior treatment of muscles from old AL rats with AICAR (an AMPK activator) elevates GU, we will test the efficacy of CR plus AICAR to optimally enhance insulin-mediated GU in muscles from old rats. These unique approaches will provide groundbreaking insights into fundamental mechanisms underlying CR- improved insulin sensitivity in muscle of older individuals.